Therapeutic Effects of Non-Euphorigenic Cannabis Extracts in Osteoarthritis.

Introduction: Osteoarthritis (OA) is disabling and degenerative disease of the joints that is clinically characterized by pain and loss of function. With no disease-modifying treatment available, current therapies aim at pain management but are of limited efficacy. Cannabis products, specifically cannabinoids, are widely used to control pain and inflammation in many diseases with no scientific evidence demonstrating their efficacy in OA. Objective: We investigated the effects of non-euphorigenic cannabis extracts, CBD oil and cannabigerol oil (CBG oil), on pain and disease progression in OA mice. Methods and Results: Twelve-week-old male C57BL/6J mice received either sham or destabilization of the medial meniscus (DMM) surgery. DMM mice were treated with vehicle, CBD oil, or CBG oil. The gait of DMM mice was impaired as early as 2 weeks following surgery and continued deteriorating until week 8, which was restored by CBD oil and CBG oil treatments throughout the disease course. Mechanical allodynia developed in DMM mice, however, was not ameliorated by any of the treatments. On the other hand, both CBD oil and CBG oil ameliorated cold allodynia. In open field test, both oil treatments normalized changes in the locomotor activity of DMM mice. CBD oil and CBG oil treatments significantly reduced synovitis in DMM mice. Only CBG oil reduced cartilage degeneration, chondrocyte loss, and matrix metalloproteinase 13 expression, with a significant increase in the number of anabolic chondrocytes. Subchondral bone remodeling found in vehicle-treated DMM mice was not ameliorated by either CBD or CBG oil. Conclusions: Our results show evidence for the therapeutic efficacy of CBD oil and CBG oil, where both oils ameliorate pain and inflammation, and improve gait and locomotor activity in OA mice, representing clinical pain and function. Importantly, only CBG oil is chondroprotective, which may provide superior efficacy in future studies in OA patients.

Early Gßγ-GRK2 Inhibition Ameliorates Osteoarthritis Development by Simultaneous Anti-Inflammatory and Chondroprotective Effects.

The G-protein-coupled receptor kinase 2 (GRK2) is an important regulator of inflammation and pathological macrophage phenotype in a variety of diseases. We hypothesize that Gßγ-GRK2 signaling promotes the early inflammatory response and chondrocyte loss in osteoarthritis (OA). Using the destabilization of the medial meniscus (DMM) model in 12-week-old male C57BL/6 mice, we determined the role of Gßγ-GRK2 signaling in synovitis, macrophage activation, and OA development. We achieved Gßγ-GRK2 inhibition at the time of DMM by administering the Gßγ inhibitor "gallein" and the GRK2 inhibitor "paroxetine" daily, starting from 2 days before DMM surgery, for a duration of 1 or 12 weeks. Synovial and cartilage structural changes were evaluated by histomorphometry, and molecular events and macrophage activation were examined. We studied the direct role of Gßγ-GRK2 in synovitis and macrophage activation in vitro using SW982 and THP1 cells. Continuous Gßγ-GRK2 inhibition initiated at the time of DMM attenuated OA development and decreased chondrocyte loss more effectively than delayed treatment. GRK2 expression and the M1 macrophage phenotype were elevated in the inflamed synovium, while early gallein and paroxetine treatment for 1 and 12 weeks following DMM resulted in their reduction and an upregulated M2 macrophage phenotype. In vitro experiments showed that Gßγ-GRK2 inhibition attenuated synoviocyte inflammation and the M1 phenotype. We show that early Gßγ-GRK2 inhibition is of higher therapeutic efficacy in OA than delayed inhibition, as it prevents OA development by inhibiting the early inflammatory response.

Aspiration-assisted bioprinting of the osteochondral interface.

Osteochondral defects contain damage to both the articular cartilage and underlying subchon- dral bone, which remains a significant challenge in orthopedic surgery. Layered structure of bone, cartilage and the bone-cartilage interface must be taken into account in the case of biofabrication of the osteochondral (OC) interface. In this study, a dual layered OC interface was bioprinted using a newly developed aspiration-assisted bioprinting (AAB) technique, which has been the first time that scaffold-free bioprinting was applied to OC interface engineering. Tissue spheroids, made of human adipose-derived stem cells (ADSCs), were differentiated in three dimensions (3D) into chondrogenic and osteogenic spheroids, which were confirmed by immunostaining and histology qualitatively, and biochemistry assays and gene expression, quantitatively. Remarkably, the OC interface was bioprinted by accurate positioning of a layer of osteogenic spheroids onto a sacrificial alginate support followed by another layer of chondrogenic spheroids overlaid by the same support. Spheroids in individual zones fused and the maintenance of phenotypes in both zones confirmed the successful biofabrication of the histomorphologically-relevant OC interface. The biofabrication of OC tissue model without the use of polymeric scaffolds unveils great potential not only in regenerative medicine but also in drug testing and disease modeling for osteoarthritis.

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

Small molecule disruption of G protein ßγ subunit signaling reprograms human macrophage phenotype and prevents autoimmune myocarditis in rats.

The purpose of this study was to determine whether blocking of G protein ßγ (Gßγ) signaling halts heart failure (HF) progression by macrophage phenotype manipulation. Cardiac Gßγ signaling plays a crucial role in HF pathogenesis. Previous data suggested that inhibiting Gßγ signaling reprograms T helper cell 1 (Th1) and Th2 cytokines, suggesting that Gßγ might be a useful drug target for treating HF. We investigated the efficacy of a small molecule Gßγ inhibitor, gallein, in a clinically relevant, experimental autoimmune myocarditis (EAM) model of HF as well as in human macrophage phenotypes in vitro. In the myocardium of HF patients, we observed that G protein coupled receptor kinase (GRK)2 levels were down-regulated compared with healthy controls. In rat EAM, treatment with gallein effectively improved survival and cardiac function, suppressed cardiac remodeling, and further attenuated myocardial protein expression of GRK2 as well as high mobility group box (HMGB)1 and its cascade signaling proteins. Furthermore, gallein effectively inhibited M1 polarization and promoted M2 polarization in vivo in the EAM heart and in vitro in human monocyte-derived macrophages. Taken together, these data suggest that the small molecule Gßγ inhibitor, gallein, could be an important pharmacologic therapy for HF as it can switch the phenotypic reprogramming from M1 to M2 phenotype in a rat model of EAM heart and in human macrophages.

Targeting the gut microbiome to treat the osteoarthritis of obesity.

Obesity is a risk factor for osteoarthritis (OA), the greatest cause of disability in the US. The impact of obesity on OA is driven by systemic inflammation, and increased systemic inflammation is now understood to be caused by gut microbiome dysbiosis. Oligofructose, a nondigestible prebiotic fiber, can restore a lean gut microbial community profile in the context of obesity, suggesting a potentially novel approach to treat the OA of obesity. Here, we report that - compared with the lean murine gut - obesity is associated with loss of beneficial Bifidobacteria, while key proinflammatory species gain in abundance. A downstream systemic inflammatory signature culminates with macrophage migration to the synovium and accelerated knee OA. Oligofructose supplementation restores the lean gut microbiome in obese mice, in part, by supporting key commensal microflora, particularly Bifidobacterium pseudolongum. This is associated with reduced inflammation in the colon, circulation, and knee and protection from OA. This observation of a gut microbiome-OA connection sets the stage for discovery of potentially new OA therapeutics involving strategic manipulation of specific microbial species inhabiting the intestinal space.

Simultaneous adrenal and cardiac g-protein-coupled receptor-gßγ inhibition halts heart failure progression.

OBJECTIVES: The authors propose simultaneous inhibition of Gßγ signaling in the heart and the adrenal gland as a novel therapeutic approach for heart failure (HF). BACKGROUND: Elevated sympathetic nervous system activity is a salient characteristic of HF progression. It causes pathologic desensitization of ß-adrenergic receptors (ß-AR), facilitated predominantly through Gßγ-mediated signaling. The adrenal glands are key contributors to the chronically elevated plasma catecholamine levels observed in HF, where adrenal α2-AR feedback inhibitory function is impaired also through Gßγ-mediated signaling. METHODS: We investigated the efficacy of a small molecule Gßγ inhibitor, gallein, in a clinically relevant, pressure-overload model of HF. RESULTS: Daily gallein treatment (10 mg/kg/day), initiated 4 weeks after transverse aortic constriction, improved survival and cardiac function and attenuated cardiac remodeling. Mechanistically, gallein restored ß-AR membrane density in cardiomyocytes, attenuated Gßγ-mediated G-protein-coupled receptor kinase 2-phosphoinositide 3-kinase γ membrane recruitment, and reduced Akt (protein kinase B) and glycogen synthase kinase 3ß phosphorylation. Gallein also reduced circulating plasma catecholamine levels and catecholamine production in isolated mouse adrenal glands by restoring adrenal α2-AR feedback inhibition. In human adrenal endocrine tumors (pheochromocytoma), gallein attenuated catecholamine secretion, as well as G-protein-coupled receptor kinase 2 expression and membrane translocation. CONCLUSIONS: These data suggest small molecule Gßγ inhibition as a systemic pharmacologic therapy for HF by simultaneously normalizing pathologic adrenergic/Gßγ signaling in both the heart and the adrenal gland. Our data also suggest important endocrine/cardiovascular interactions and a possible role for small molecule Gßγ inhibition in treating endocrine tumors such as pheochromocytoma, in addition to HF.

A novel phenylpyridazinone, T-3999, reduces the progression of autoimmune myocarditis to dilated cardiomyopathy.

Regardless of the origin, injury to the heart can result in cardiomyocyte hypertrophy, fibrosis, and cell death. Myocarditis often progresses to dilated cardiomyopathy (DCM), a major cause of heart failure. In our study, we used a rat model of myosin-induced experimental autoimmune myocarditis (EAM), in which the heart transits from an acute phase (inflammatory myocarditis) to a chronic phase (remodeling and DCM). Our objective was to investigate whether T-3999, a novel phenylpyridazinone, can reduce this progression. Four weeks after myosin injection, T-3999 was administered daily to male Lewis rats in two doses (3 and 10 mg/kg, orally). Four weeks later, treatment was terminated; hemodynamic and echocardiographic measurements were performed; hearts were excised for histopathology and estimation of histamine, mRNA, and protein levels. Mortality rate was reduced by drug treatment. T-3999 reduced % fibrosis and tissue collagen III. Profibrotic markers-transforming growth factor-ß(1), tumor necrosis factor-α, and galectin-3--were attenuated by treatment. Mast cell density and degranulation, and tissue histamine concentration were also reduced. This indicates an anti-inflammatory effect of the drug in reducing fibrosis. Hypertrophy was reduced as reflected by reduced myocyte diameter and natriuretic peptide expression. T-3999 treatment increased the sarcoendoplasmic reticulum Ca(2+) ATPase 2 protein level and improved several cardiac function parameters. The reduction of the remodeling process and improvement in myocardial function suggest an effect of T-3999 in attenuating ventricular remodeling in post-myocarditis DCM.

Local delivery of angiotensin II receptor blockers into the kidney passively attenuates inflammatory reactions during the early phases of streptozotocin-induced diabetic nephropathy through inhibition of calpain activity.

BACKGROUND/AIMS: Inhibition of the renin-angiotensin-aldosterone system plays a pivotal role in the prevention and treatment of diabetic nephropathy. Angiotensin II receptor blockers (ARB) exert a renoprotective effect and attenuate the progression of diabetic nephropathy. However, the underlying cellular and molecular mechanisms in the kidney remain to be elucidated. The present study was undertaken to focus on the effect of local angiotensin II type 1 receptor blockade on the inflammatory reaction during the early stages of diabetic nephropathy. METHODS AND RESULTS: Local ARB treatment significantly reduced urinary protein excretion and serum blood urea nitrogen levels in streptozotocin-induced diabetic nephropathy. In addition, this treatment attenuated monocyte/macrophage infiltration into the glomeruli and the enhanced glomerular expression of endothelial nitric oxide synthase at both the mRNA and protein levels. Immunohistochemical study revealed activation of nuclear factor (NF)-kappaB, as shown by an increase in the expression of the p65 subunit of NF-kappaB and its translocation from the cytoplasm to the nucleus in both tubular epithelial and glomerular cells of the diabetic kidney. Local ARB treatment induced an apparent reduction in p65 nuclear localization and intensity of staining. To search for a common and fundamental candidate that influences endothelial cell function and vascular inflammation, we examined glomerular calpain activity in diabetic rats with or without ARB treatment. Glomerular expression of 145/150-kDa spectrin breakdown products, a specific product of calpain activation, was dramatically increased in diabetic animals while the protein expression reverted to a normal level after ARB treatment. CONCLUSION: Our findings provide a conceptual basis for the development of therapeutic strategies aiming at local inhibition of the renin-angiotensin system to prevent the progression of diabetic nephropathy.

Chymase inhibition reduces the progression to heart failure after autoimmune myocarditis in rats.

Chymase has been known as a local angiotensin II-generating enzyme in the cardiovascular system in dogs, monkeys, hamsters, and humans; however, recently it was reported that chymase also has various other functions. Therefore, we decided to examine whether the inhibition of chymase improves disease conditions associated with the pathophysiology of dilated cardiomyopathy in rats and its possible mechanism of action as rat chymase is unable to produce angiotensin II. We examined the effect of TY-51469, a novel chymase inhibitor (0.1 mg/kg/day [group CYI-0.1, n = 15] and 1 mg/kg/day [group CYI-1, n = 15]), in myosin-immunized postmyocarditis rats. Another group of myosin-immunized rats was treated with vehicle (group V, n = 15). Age-matched normal rats without immunization (group N, n = 10) were also included in the study. After 4 weeks of treatment, we evaluated cardiac function; area of fibrosis; fibrogenesis; levels of transforming growth factor (TGF)-beta1 and collagen III; hypertrophy and its marker, atrial natriuretic peptide (ANP); and mast cell activity. Survival rate and myocardial functions improved dose-dependently with chymase inhibitor treatment after myosin immunization. A reduction in the percent area of myocardial fibrosis, fibrogenesis, myocardial hypertrophy, and mast cell activity along with a reduction in TGF-beta1, collagen III, and ANP levels in the myocardium were noted in postmyocarditis rats that received chymase inhibitor treatment. The treatment also decreased myocardial aldosterone synthase levels in those animals. Inhibition of chymase reduces the pathogenesis of postmyocarditis dilated cardiomyopathy and progression to heart failure by preventing the pathological remodeling and residual inflammation in rats.

Comparative effects of pranidipine with amlodipine in rats with heart failure.

The aim of the present study was to compare the cardioprotective properties of long-acting calcium channel antagonist pranidipine with amlodipine in rat model of heart failure induced by autoimmune myocarditis. Twenty-eight days after immunization the surviving rats were randomized for the oral administration of low-dose amlodipine (1 mg/kg/day), high-dose amlodipine (5 mg/kg/day), pranidipine (0.3 mg/kg/day) or vehicle (0.5% methylcellulose). After oral administration for 1 month, the animals underwent echocardiography and hemodynamic analysis. Histopathology, immunohistochemistry, and Western immunoblotting were carried out in the heart samples. Both pranidipine and high-dose amlodipine increased survival rate. Although the heart rate did not differ among the four groups, left ventricular end-diastolic pressure was significantly decreased and +/-dP/dt was increased in the pranidipine- and high-dose amlodipine-treated rats, but not in low-dose amlodipine-treated rats. In comparison to amlodipine treatment, pranidipine treatment significantly reduced myocyte size and central venous pressure. Furthermore, both pranidipine and high-dose amlodipine treatment significantly reduced myocardial protein levels of atrial natriuretic peptide and inducible nitric oxide synthase, whereas pranidipine only significantly decreased tumor necrosis factor-alpha, and improved sarcoplasmic reticulum Ca2+ ATPase2 protein levels. We conclude that pranidipine ameliorates the progression of left ventricular dysfunction and cardiac remodeling in rats with heart failure after autoimmune myocarditis in a lower dose when compared to amlodipine and which may be a clinically potential therapeutic agent for the treatment of heart failure.

Effects of angiotensin II receptor blocker (candesartan) in daunorubicin-induced cardiomyopathic rats.

BACKGROUND: Daunorubicin is an anthracycline anti-tumor agent; anthracycline chemotherapy in cancer can cause severe cardiomyopathy leading to a frequently fatal congestive heart failure; the first-line treatment is diuretics and digoxin. Recently, angiotensin-converting enzyme inhibitors have been shown to be effective in the treatment of such toxicity. The purpose of this study was to investigate the effects of angiotensin II type-1 receptor antagonist (candesartan) in a rat model of daunorubicin-induced cardiomyopathy. METHODS: Rats were treated with a cumulative dose of 9 mg/kg body weight daunorubicin (i.v.). 28 days later, after the development of cardiomyopathy, animals were randomly assigned to candesartan-treated (5 mg/kg/day, p.o.) or vehicle-treated groups; age-matched normal rats were used as the control group. Candesartan treatment was continued for 28 days. Hemodynamic and echocardiographic parameters were measured, cardiac protein and mRNA were analyzed, and histopathological analyses of myocardial fibrosis, cell size and apoptosis were conducted. RESULTS: Following cardiomyopathy, left ventricular end diastolic pressure and left ventricular systolic dimension were significantly elevated; while % fractional shortening and Doppler E/A ratio were significantly reduced. Cardiomyopathic hearts showed significant increases in % fibrosis, % apoptosis, and myocyte diameter/body weight ratio; candesartan treatment reversed these changes. Fas-L protein overexpression in myopathic hearts was significantly suppressed by treatment with candesartan. Moreover, SERCA2 mRNA and protein expression were both down-regulated in myopathic hearts and restored to normal by candesartan treatment, significantly. CONCLUSIONS: Our findings suggest that candesartan treatment significantly improved the left ventricular function and reversed the myocardial pathological changes investigated in this model of daunorubicin-induced cardiomyopathy; suggesting its potentials in limiting daunorubicin cardiotoxicity.